PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) remains one of the major causes of morbidity in humans. The only available vaccine against tuberculosis (TB), BCG, is ineffective in protecting against adult pulmonary TB. This warrants the development of alternative vaccines or booster strategies that confer better protection against Mtb. Several subunit vaccine candidates have been developed using Mtb protein antigens that elicit potent Th1 responses. However, little is known about the potential use of Mtb lipid antigens in vaccines against Mtb. Since the Mtb cell wall is lipid rich, with mycolic acid (MA) being the most abundant lipid species, harnessing cellular immune responses to MA could be a promising vaccine strategy. CD1 molecules present a range of Mtb lipids to cognate T cells. They are divided into group 1 (CD1a, CD1b and CD1c) and group 2 (CD1d). Of the four CD1 isoforms, CD1b presents the largest cohort of Mtb-derived lipids, including MA. MA-specific CD1b- restricted T cells have been detected in the blood as well as disease sites of Mtb-infected individuals. In addition, we have recently shown that MA-specific CD1b-restricted T cells play a protective role during Mtb infection in a humanized transgenic mouse model. These data suggest that MA would be an ideal model antigen to test the feasibility of a lipid-based subunit vaccine to TB. In this proposal, we will investigate the possibility of using nanoparticle-encapsulated MA as a subunit vaccine to examine its protective capacity in human CD1 transgenic mice challenged with Mtb. As suggested by our preliminary data, use of nanoparticles facilitates easy delivery of MA to the lungs through the intranasal route. Since it is known that the size of nanoparticles affects antigen-presenting cell (APC) targeting, in specific aim 1, we propose to compare the efficiency of antigen presentation in vivo when either micelles (~30 nm diameter) or polymersomes (~120 nm diameter) are used to encapsulate MA together with the dual adjuvants, CpG ODN and MPLA. In an attempt to elicit the most robust MA-specific T cell response upon immunization, we will further test if the co-delivery of ?-GalCer and GM-CSF will enhance the immunogenicity of these MA nanoparticles. The best combination of MA-nanoparticles and adjuvants from specific aim 1 will be used in specific aim 2 to immunize human CD1 transgenic mice to induce MA-specific T cell responses in a polyclonal setting. Lastly, the protective capacity of vaccination with MA-adjuvant nanoparticles against Mtb infection will be evaluated. Collectively, this study will not only provide a ?proof of concept? that group 1 CD1-restricted lipid antigens can be included in subunit vaccines against Mtb infection but also explore the possibility of using nanoparticles for efficient lipid antigen delivery to the primary site of infection.